DE3751468T2 - METHOD FOR PRODUCING OLIGONUCLEOTIDES AND COMPOUNDS FOR FORMING HIGH-MOLECULAR PROTECTION GROUPS. - Google Patents

Description

TECHNICAL AREA

The present invention relates to a method for synthesizing an oligonucleotide having a chain extended according to the bond in nucleic acid by condensing a nucleotide or oligonucleotide. More specifically, the present invention relates to a method for synthesizing a chain extended oligonucleotide by condensing a nucleotide or oligonucleotide having a high molecular weight protecting group introduced therein, in which the condensation reaction can be carried out in the homogeneous system and the separation and purification can be carried out in the heterogeneous system and a product of high purity can be obtained at a high space-time yield.

Furthermore, the present invention relates to a compound for forming a polymeric protecting group useful for the synthesis of the above-mentioned oligonucleotide and for the synthesis of nucleic acid, oligosaccharides and polysaccharides.

BACKGROUND KNOWLEDGE

Nucleic acid has a structure in which many hydroxyl groups at specific sites of the saccharide component of the nucleotide are bonded via a diester group of phosphoric acid, that is, a structure in which alcoholic hydroxyl groups of nucleotide and phosphoric acid groups are sequentially bonded via ester bridges. Accordingly, the following two methods for condensing two nucleotides can be selected according to the bond in nucleic acid:

(a) the process in which a 3'- or 2'-phosphoric acid group is condensed with a 5'-hydroxyl group;

(b) the process in which a 3'- or 2'-hydroxyl group is condensed with a 5'-phosphoric acid group.

Since the phosphoric acid group is bulky and the 5-hydroxyl group consists of a primary alcohol and has a relatively high degree of freedom and since the 3ro - and 2'-hydroxyl groups consist of a secondary alcohol and are close to each other, process (a) is the most advantageous of the two processes mentioned above.

The process (a) is further divided into the diester process, the triester process, the phosphite process and the H-phosphonate process according to the type of condensation reaction. In view of the reaction yield, the stability of the intermediate, the reaction rate and the ease of purification of the product, the triester process and the phosphite process are considered to be relatively advantageous.

The reaction of the triester process can be expressed by the following formula (1): condensing agent

In formula (1), B₁ and B₂ represent a base moiety in which the amino group is protected, R₁ represents a protecting group for the hydroxyl group, R₂ and R₄ represent hydrogen in the case of deoxyribonucleotide or a protected hydroxyl group in the case of ribonucleotide, and R₃ and R₅ represent a protecting group for the phosphoric acid group.

After the above-mentioned reaction to form triester, the protective groups for the functional groups are removed to obtain the desired product. In general, there is no great difference in the solubility or the like of the reaction product, unreacted substance and by-product, and therefore it is not always easy to separate and purify them.

To facilitate this separation and purification, several methods have been proposed to bring about differences in the physical properties of the reaction products by using high molecular weight compounds as the compound forming a protecting group for the functional group. If the compound having a polymeric protecting group is solid under the reaction conditions or is available as a solid after the reaction, the desired product can be easily separated and retained by filtration and washing.

The site for forming R5 in formula (1) is considered to be the site to which the polymeric protecting group is bonded, and the use of a cross-linked polystyrene or silica gel having its functional group bonded thereto as R5 has been reported [V.A. Efimov et al., Nucleic Acid Res. 11, 8369 (1983)]. The oligonucleotide formed by bonding a polymeric protecting group as mentioned above to the terminal phosphoric acid group is solid, and a number of solid-phase processing techniques have been established. In contrast, the method in which the condensation reaction represented by formula (1) is carried out in a homogeneous solution is called the liquid-phase method. In conjunction with the diester method, the use of polyvinyl alcohol [H. Scott et al., Macromolecular Chemistry, 173, 247 (1973)], polyethylene glycol [H. Kosler, Tetrahedron Letters, No. 16, 1535 (1972)], polystyrene [H. Hayatsu, H. G. Kohorana, J. Am. Chem. Soc., 88 3182 (1966)] and vinyl alcohol/polyvinylpyrrolidone copolymer [H. Seliger, G. Aumamann, Tetrahedron Letters, No. 31, 2911(1973)] have been suggested as the compound for forming a polymeric protecting group. When these solvent-soluble polymers are used, the process is considered as a liquid phase process, but in this case it is not easy to separate and purify the reaction product.

The present invention intends to provide a process for synthesizing a chain-extended oligonucleotide according to the triester method or the phosphite method, in which separation and purification of the desired product can be easily carried out and the desired product with high purity can be obtained in a high yield.

According to the present invention, there is provided a process for synthesizing a chain-extended oligonucleotide, which comprises condensing a mononucleotide, an oligonucleotide, a mononucleotide succinate or an oligonucleotide succinate in a homogeneous system in a solvent, wherein only the terminal 5'-hydroxyl group is not blocked and the other functional groups are blocked by a plurality of low molecular weight protecting groups and a protecting group containing a hydroxyl group, with a mononucleotide or oligonucleotide in which the functional groups other than the terminal phosphoric acid group are blocked by low molecular weight protecting groups, characterized in that the protecting group containing the hydroxyl group is a polysaccharide derivative which is controllably soluble in the solvent, wherein the compound forming the protecting group from the polysaccharide derivative is formed by attaching a spacer and a functional group to a polysaccharide derivative.

Further, according to the present invention, there is provided a compound for forming a protective group from the polysaccharide derivative, which is represented by the following general formula (I):

where C�6H�7O₂ is an anhydrous glucose residue, R is

n is an integer between 10 and 2,000, x is a number between 0.4 and 0.8, and y is a number between 1.0 and 2.0.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a chromatogram obtained by reverse-phase high performance liquid chromatography of an oligonucleotide (RNA type undecamer) from which the protecting groups were completely removed obtained in an example (Example 9) of the present invention;

Fig. 2 is a chromatogram obtained by re-chromatographing a purified product of the oligonucleotide shown in Fig. 1;

Fig. 3 is a chromatogram obtained by reverse-phase high performance liquid chromatography of an oligonucleotide (DNA type octamer) from which the protecting groups were completely removed, obtained in an example (Example 15) of the present invention; and

Fig. 4 is a chromatogram obtained by re-chromatographing a purified product of the oligonucleotide shown in Fig. 3;

BEST MODE FOR CARRYING OUT THE INVENTION

The compound for forming a protective group from a polysaccharide derivative as used in the present invention is soluble in a conventional solvent for an oligonucleotide and an oligonucleotide blocked by a low molecular weight protective group, and when a non-solvent is added to a solution in this conventional solvent, the compound can be easily precipitated and separated. Compounds for forming a protective group from a polysaccharide derivative suitable for achieving the object of the present invention can be derived from polysaccharides. Protective group-forming compounds produced by using acetyl cellulose having a low degree of acetylation, a mixed cellulose ester composed of acetyl cellulose and propionyl cellulose, or a mixed cellulose ester composed of acetyl cellulose and butyryl cellulose are particularly useful. To prepare a protecting group-forming compound by modifying a polysaccharide derivative, for example acetylcellulose, a hydroxyethylsulfonyl group is preferably introduced as the functional group which blocks the phosphoric acid group. The -OH group of the hydroxyethylsulfonyl group blocks the phosphoric acid group by forming a phosphate ester.

The skeleton of the compound that generates a protecting group from a polysaccharide derivative forms a spatial obstacle by reacting with a bulky compound such as a nucleotide, and therefore, in order to impart sufficient reactivity to the hydroxyethylsulfonyl group, the functional group must be removed from the polymeric main chain by a certain distance. Specifically, the compound which forms a protective group from a polysaccharide derivative and is preferably used in the process of the present invention is an acetylcellulose derivative in which an ester bond is formed between the hydroxyl group of the main chain and, for example, a 4-(2-hydroxyethylsulfonyl)dihydrocinnamoyl group. Acetylcellulose having a degree of substitution of about 1.7 to about 1.8, which is preferred as the starting material for the protective group-forming compound, is characterized in that the number of hydroxyl groups participating in the ester-forming reaction is large, that the solubility is good and that the compound is soluble in many polar solvents.

The reaction for the synthesis of the above-mentioned acetylcellulose derivative is represented by the following formulas (2) and (3).

In formula (2), MMTr represents a monomethoxytrityl group. The reaction formula (2) represents the synthesis of 4-(2-monomethoxytrityloxyethylthio)dihydrocinnamic acid < 3 > by the dehydrochlorination condensation reaction between a monomethoxytrityl ether < 1 > of ethylene chlorohydrin and 4-mercaptodihydrocinnamic acid < 2 >. < 3 > + Acetylcellulose 1-Meim Pyridine Acetylcellulose

In formula (3), Tps stands for 2,4,6-triisopropylbenzenesulfonyl chloride and 1-Meim stands for 1-methylimidazole.

Formula (3) represents the reaction process in which 4-(2-monomethoxytrityloxyethylthio)dihydrocinnamic acid <3> and acetyl cellulose are subjected to ester condensation using 1-methylimidazole as the catalyst and 2,4,6-triisopropylbenzenesulfonyl chloride as the condensing agent, unreacted hydroxyl groups in the acetyl cellulose are esterified by acetic anhydride, oxidation is effected by using hydrogen peroxide and sodium tungstate to introduce sulfonyl groups into the molecule, and a polymer-supported compound <4> is synthesized by removing the monomethoxytrityl group using zinc bromide. The compound <4> is an acetyl cellulose derivative having a 4-(2-hydroxyethylsulfonyl)dihydrocinnamoyl group. This compound is soluble in pyridine.

For ease of explanation, the present invention will now be described with reference to the condensation reaction between a mononucleotide and a mononucleotide, however, the techniques described below can also be applied to embodiments of the present invention in which one or both of the reactants are oligonucleotides. Briefly, according to the present invention, the compound <4> is reacted with a nucleotide in which the functional groups are blocked except for a single phosphoric acid group, for example, 5'-O-dimethoxytrityl-2'-O-tetrahydropyranylribonucleoside 3'-(2-chlorophenyl)phosphate <5> to block the phosphoric acid group, as shown by the following formula (4) is shown: 1-Meim Extraction Pyridine Acetylcellulose

In formula (4), DMTr represents a dimethoxytrityl group, Thp represents a tetrahydropyranyl group, PhCl represents a 2-chlorophenyl group and TsOH represents p-toluenesulfonic acid.

Between < 4 > and < 5 >, cold water is added to the condensation reaction liquid to stop the reaction, and when the reaction liquid is extracted with methylene chloride, the reaction product is transferred to the methylene chloride layer. The hydroxyl group of the hydroxyethyl group in < 4 >, which did not participate in the reaction, is blocked by an acetyl group, and p-toluenesulfonic acid is added to release the dimethoxytrityl group. When a non-solvent, that is, ethanol in this case, is added to the reaction liquid, < 6 > is obtained as the precipitate. The compound < 6 > is a mononucleotide blocked by the protective group from the polysaccharide derivative.

Then, < 5 > and < 6 > are condensed to link two nucleotides via a triester of phosphoric acid to obtain a compound < 7 > in which the terminal phosphoric acid group is blocked by the polymeric protecting group, as shown by the following formula (5): l-Meim Pyridine Acetylcellulose Et₃N-Pyridine

The protective group from the polysaccharide derivative which blocks the terminal phosphoric acid group of <7> is isolated, and the product is separated to obtain the objective dinucleotide derivative <8>. This isolation reaction is a β-elimination reaction, and an acetylcellulose derivative <9> having a terminal olefin group is formed. The derivative <9> is precipitated by adding ethanol to the reaction liquid and can be easily separated from <8>. The separation of <8> from the unreacted nucleotide and the like is achieved by reverse phase chromatography.

The oligonucleotide thus synthesized is used in the state in which the protecting group bonded thereto serves as the starting material for the chain-extending reaction. On the other hand, in order to obtain the oligonucleotide from the oligonucleotide having the protecting group bonded thereto, the protecting group is removed in the following manner. First, treatment is carried out for 16 hours in 0.5 M 2-pyridine aldoximate - 0.5 M 1,1,3,3-tetramethylguanidine/pyridine - water (9:1 v/v) at room temperature to remove the 2-chlorophenyl group as the group protecting the phosphoric acid group, and then treatment is carried out with aqueous ammonia (28% to 30%) at 50°C for 5 hours to remove the protecting group for the amino group of the base portion.

Further, treatment with an aqueous hydrochloric acid solution having a pH of 2 at room temperature for one day is carried out to remove the dimethoxytrityl group as the protective group for the 5'-hydroxyl group and the tetrahydropyranyl group as the protective group for the 2'-hydroxyl group.

An oligonucleotide from which the protecting groups are completely removed is obtained according to the above-mentioned procedures.

According to the method of the present invention, optional oligonucleotides ranging from a nucleotide dimer to a nucleotide polymer (for example, an eicosamer) can be obtained. Specifically, an oligonucleotide is obtained from a mononucleotide, a high molecular weight oligonucleotide is obtained by condensing this oligonucleotide with a mononucleotide or oligonucleotide, and an optional polymer is obtained by repeating these procedures.

The synthesis of the chain-extended oligonucleotide has been described in detail with reference to the triester method. Nevertheless, the method of the present invention can be similarly applied to the phosphite method.

Polysaccharides, oligosaccharides and nucleic acid, which are important as physiologically active substances, are generally formed by condensing a small number of kinds of monosaccharides or mononucleotides with a certain regularity, and pure synthesis of them is chemically very important, but contains many unsolved problems. Most of the problems encountered in the synthesis arise because it is difficult to selectively carry out the reaction for converting the components having polyfunctional groups into a desired bonding form. Furthermore, since there is no great difference in the physical properties of the starting material, the target product and the by-product, it is very difficult to separate and purify the target product even if the target reaction is well developed. For these reasons, the technique of selectively protecting each functional group of the components and isolating the protecting group provides an important tool for the synthesis or analysis of polysaccharides, oligosaccharides and nucleic acid.

The present invention is characterized in that the protective group for the terminal phosphoric acid group of the nucleotide is formed from a polymeric compound which is soluble in solvents and can be easily separated by precipitation, that the condensation reaction is carried out in a homogeneous solution phase and that the product can be recovered by solid-liquid separation.

The characteristics of the present invention will now be described by comparing the process of the present invention with the conventional processes.

I) Comparison with a liquid phase process

The main points of the conventional liquid phase process are represented by the following formulas (6) and (7). Phosphorylation Silica gel column chromatography Removal of the group R₁

In formula (6), R6 represents -H or OR7, and Rg represents -OR5 or -NHR9. Condensation Silica gel column chromatography Group removal Reversed phase silica gel chromatography

In the liquid phase process, that is, the process using only low molecular weight protecting groups, the products obtained in the respective steps must be capable of being purified by column chromatography. In contrast, the process of the present invention is advantageous in that the product can be purified by precipitating the product in the form of a polymer derivative by a non-solvent can be easily and rapidly purified and that an excess amount of the starting material not bound to the polymer is used and the excess part of the starting material is retained with high purity.

II) Comparison with a solid phase method

In all conventional solid phase methods, the reaction is carried out in a heterogeneous system. Accordingly, the amount supported on the solid phase carrier, that is, the amount of the starting material bound, is small. Even in the case of a polystyrene resin crosslinked with 1% to 2% of divinylbenzene, which is a conventional carrier considered to have a high supported amount, the supported amount of a nucleoside or nucleotide is 0.1 millimol/g to 0.4 millimol/g. In contrast, in the case of acetylcellulose used in the present invention, a large amount of the nucleotide can be supported because the amount of the spacer incorporated is 1.65 millimol/g to 1.97 millimol/g and the condensation reaction for introducing a nucleotide is significantly improved in quantity. Furthermore, in the conventional solid phase process, since the reaction is a heterogeneous system, it is necessary to use the condensing agent, the nucleotide or oligonucleotide 3'-phosphodiester derivative, in large excess, while in the process of the present invention, since the condensation reaction is carried out in a homogeneous system, the amounts of the starting materials can be reduced and the space-time yield can be increased. In specific examples of the present invention, acetyl cellulose having a low degree of acetylation (D.S. = 1.7 to 1.8) is used as the starting material of the compound forming a protective group from a polysaccharide derivative, and acetyl cellulose having a low degree of acetylation is considered advantageous in that the acetyl cellulose has many hydroxyl groups, the acetyl cellulose is soluble in pyridine, pyridine/water and the like, hydroxyl groups not participating in the reaction can be easily blocked, and the acetyl cellulose has suitable nonsolvents.

Another characteristic feature of the compound used in specific examples of the present invention which forms a protective group from the polysaccharide derivative is that a spacer is introduced to bond functional groups to the acetyl cellulose. In specific examples of the present invention, in the reaction for introducing a functional group, 4-(2-monomethoxytrityloxyethylthio)dihydroxycinnamic acid <3> is reacted with a spacer. This reaction is an esterification, and the reaction amount per unit amount of the polymer is large, and the amount of the spacer introduced is large. This dominates the bonded amount of the functional group for blocking the phosphoric acid group of the nucleotide, and as a result, a large amount of bonded nucleotide is obtained per unit amount of the polymer. In specific examples of the present invention, a 2-hydroxyethylsulfonyl group is used as the functional group for blocking the phosphoric acid group, and since the alcoholic hydroxyl group in this functional group is a primary hydroxyl group, the phosphoric acid esterification can be easily achieved. Furthermore, since the sulfonyl group is located adjacent to the carbon atom at the β-position, the functional group can be removed by β-elimination under mild conditions that have no influence on other functional groups. This sulfonyl group is obtained by post-oxidation of the sulfur atom of 4-(2-monomethoxytrityloxyethylthio)dihydroxycinnamic acid < 3 >. This sulfur atom can be used to determine the spacer introduced.

The alcoholic hydroxyl group of the 2-hydroxyethylsulfonyl group of the compound used in the present invention, which forms the protective group of the polysaccharide derivative, reacts with a carboxylic acid or sulfonic acid as well as with phosphoric acid to form an ester bond and act as a protective group. In the present invention, in addition to the reaction with the phosphoric acid group, a reaction in which succinic acid is bonded to the hydroxyl group of the 2-hydroxyethylsulfonyl group of the polysaccharide derivative is used, and the compound which forms the protective group of the polysaccharide derivative is used as the protective group for the resulting carboxyl group-terminated mononucleotide succinate or oligonucleotide succinate.

Examples

The present invention will now be described in detail with reference to the following examples, which in no way limit the scope of the invention.

The synthesis of a compound forming a protecting group from a polysaccharide derivative is shown in Example 1, the synthesis of RNA-type oligonucleotides is shown in Examples 2 to 9, and the synthesis of DNA-type oligonucleotides is shown in Examples 10 to 15.

example 1 Synthesis of 4-(2-hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose< 4> i)2-Chloroethylmonomethoxytrityl ether < 1 >

2-Chloroethanol (4.02 mL, 60 mmol) and monomethoxytrityl chloride (9.2643 g, 30 mmol) were stirred in pyridine (150 mL) at room temperature for 3 hours. Cold water (15 mL) was added to the reaction solution, and the mixture was stirred for 30 minutes, and the reaction mixture was extracted with chloroform (500 mL) and washed with water (200 mL x 3 times). The organic layer was dried with anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration, and the filtrate was distilled under a reduced pressure. n-Hexane (100 mL) was added to the residue to cause crystallization, and thus 9.6307 g (yield = 91%) of 2-chloroethyl monomethoxytrityl ether <1> was obtained.

m.p.: 87ºC to 88ºC

¹H-n.m.r. (CDCl₃-TMS): δ3.30-3.50 (4H, m, -C-C₂ x 2), 3.60 (3H, s, OC₃), 6.70 (2H, d, J = 9 Hz, phenyl proton x 2), 6.97 - 7.53 (12H, m, phenyl proton x 12)

Elemental analysis values as C₂₂H₂₁O₂Cl:

Calculated values: C = 74, 89, H = 6.00

Values found: C = 75.00, H = 5.98

ii) 4-mercaptodihydrocinnamic acid < 2 >

4-Mercaptodihydrocinnamic acid was synthesized according to the method of Gait et al. [M.J. Gait and R.C. Sheppard, Nucleic Acids Res., 4, 1135 to 1158 (1977)].

iii) 4-(2-hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose < 4 >

2-Chloroethylmonomethoxytrityl ether <1> (2.1172 g, 6 mmol) and 4-mercaptodihydrocinnamic acid <2> (0.9112 g, 5 mmol) were dissolved in ethanol (15 mL), and diisopropylethylamine (2.45 mL, 15 mmol) was added to the solution, and the mixture was heated and refluxed for 12 hours. The solvent was removed by distillation under a reduced pressure, and the residue was purified by silica gel column chromatography to obtain 1.9945 g (yield = 80%) of 4- (2-monomethoxytrityloxyethylthio)dihydrocinnamic acid <3> in the form of a syrup.

¹H-n.m.r. (CDCl₃-TMS): δ2.56 (2H, t, J = 7.6 Hz, C-C₂), 2.83 (2H, t, J = 7.6 Hz, C-C₂), 3 ,01(2H, t, J = 6.8 Hz C-C 2 ), 3.32 (2H, t, J = 6.8 Hz, C-C 2 ), 3.64 (3H, s, OC 3 ) , 6.75 (2H, d, J = 8.8 Hz, phenyl proton x 2), 6.98 - 7.45 (16H, m, phenyl proton x 16)

iv) 4-(2-Monomethoxytrityloxyethylthio)dihydrocinnamic acid <3> (2.7924 g, 5.6 mmol) and acetylcellulose (D.S. = 1.77) (1.1794 g) were dissolved in pyridine (5 mL), and the pyridine was removed by distillation under a reduced pressure. This procedure was repeated three times. Water in the mixture was removed by azeotropic dehydration, and the residue was dissolved in pyridine (28 mL), 2,4,6-triisopropylbenzenesulfonyl chloride (4.0231 g, 14.0 mmol) and 1-methylimidazole (2.24 mL, 28.0 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours.

Cold water (5 ml) was added to the reaction solution, and the mixture was stirred for 30 minutes, extracted with chloroform and washed with water. The organic layer was concentrated under a reduced pressure. The residue was subjected to azeotropic distillation with pyridine (5 ml x 3 times) and dissolved in pyridine (14 ml), acetic anhydride (4.7 ml) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, and a small amount of toluene was added to the concentrate, and azeotropic distillation was carried out under a reduced pressure until the smell of pyridine disappeared. The residue was dissolved in dioxane (130 ml) - acetic acid (9 ml), and an aqueous solution (19 ml) of Na₂WO₄ H₂O (0.1869 g) was added to the solution. The temperature was raised to 80°C, and 30% aqueous hydrogen peroxide (28 ml) was dropped into the mixture over a period of 1 hour, and the mixture was stirred for 1 hour. The reaction temperature was lowered to room temperature, and the reaction mixture was extracted with chloroform (150 ml) - pyridine (20 ml) and washed with water (100 ml x 2 times). The organic layer was concentrated under a reduced pressure, the residue was dissolved in chloroform (30 ml), and the solution was dropped into ethanol (500 ml) with vigorous stirring. The precipitate formed was collected by filtration, washed with ethanol (200 ml) and dried under a reduced pressure to obtain 2.9753 g of a white powder.

The white powder thus obtained (2.6316 g) was dissolved in a 0.7 M solution (30 ml) of zinc bromide in chloroform - methanol (7:3 v/v), and the solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under a reduced pressure until the volume was reduced to about 1/2, and the concentrate was dropped into ethanol (400 ml) with vigorous stirring. The precipitate formed was retained by filtration, washed with ethanol (100 ml) and dried under a reduced pressure to obtain 1.8160 g of 4-(2-hydroxyethylsulfonyl)dihydrocinnamoyl acetyl cellulose <4> in the form of a white powder. The amount of spacer incorporated into compound <4> was calculated to be 1.65 mmol/g of the S content (5.30%, 1.65 mmol S per gram).

Example 2 Synthesis of the dinucleotide 3'-phosphodiester derivative < 8 > (B₁ = B₂ = N³-anisoyluracil-1-yl) using 4-(2-hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose < 4 >

4-(2-Hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose <4> (1.65 mmol/g) (0.2424 g, 0.4 mmol) and triethylamine-N3-anisoyl-5'-O-dimethoxytrityl-2'-O-tetrahydropyranyluridine-3'-(2-chlorophenyl)phosphate <5> (0.3170 g, 0.3 mmol) were dissolved in pyridine (3 mL), and the pyridine was removed by distillation under a reduced pressure. This procedure was repeated three times, and water in the mixture was removed by azeotropic dehydration. The residue was dissolved in pyridine (3 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.2726 g, 0.9 mmol) and 1-methylinudazole (0.14 mL, 1.8 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (0.5 mL) was added to the reaction solution, and the mixture was stirred for 30 minutes, and methylene chloride (50 mL) was added to the mixture, and the mixture was washed with water (30 mL). The organic layer was concentrated under a reduced pressure, and the residue was subjected to azeotropic dehydration with pyridine (3 mL x 3 times). The residue was dissolved in pyridine (7.5 mL), acetic anhydride (2.5 mL) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, a small amount of toluene was added to the concentrate, and azeotropic distillation was carried out under a reduced pressure until the pyridine odor disappeared.

The residue containing the acetylcellulose derivative < 4 >, in which a small amount Triethylamine N³-anisoyl-5'-O-dimethoxytrityl-2'-O-tetrahydropyranyluridine-3'-(2-chlorophenyl)phosphate <5> incorporated was dissolved in a small amount of methylene chloride, and the solution was dropped into ethanol to convert compound <4> into a powder (3.1 mg). The dimethoxytrityl group was removed from the 5'-hydroxyl group by treatment with a 2% solution of p-toluenesulfonic acid in chloroform-methanol (7:3 v/v), and staining was carried out in 60% perchloric acid-ethanol (3:2 v/v), and the amount of compound <5> incorporated into the acetylcellulose derivative <4> was determined by measuring the absorbance at 498 nm (ε = 72000). It was found that the amount of compound <5> introduced was 0.56 mmol/g and the condensation yield was 99%.

The residue was dissolved in chloroform-methanol (7:3 v/v) (10 ml), and the solution was cooled to 0 °C, and a solution of p-toluenesulfonic acid monohydrate (0.3954 g) in chloroform-methanol (7:3 v/v) (5 ml) was added to the above solution. The mixture was stirred for 15 minutes, and the reaction solution was neutralized with a saturated aqueous solution of sodium hydrogen carbonate and extracted with chloroform. The organic layer was concentrated under a reduced pressure, the residue was dissolved in chloroform (10 ml), and the solution was added dropwise into ethanol (200 ml) with vigorous stirring. The precipitate formed was retained by filtration to obtain 0.3330 g (0.224 mmol, yield = 75%, 0.674 mmol/g) of the powdery compound < 6 > [see formula (4)].

The compound <6> (0.3330 g) and triethylamine-N3-anisoyl-5'-O-dimethoxytrityl-2'- O-tetrahydropyranyluridine-3'-(2-chlorophenyl)phosphate <5> (0.2958 g, 0.28 mmol) were subjected to azeotropic dehydration with pyridine (3 mL x 3 times). The residue was dissolved in pyridine (2.8 mL), 2,4,6-triisopropylbenzenesulfonyl chloride (0.2544 g, 0.84 mmol) and 1-methylimidazole (0.13 mL, 0.168 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (0.5 ml) was added to the reaction liquid, and the mixture was stirred for 30 minutes, extracted with methylene chloride and washed with water. The organic layer was concentrated under a reduced pressure. A small amount of the residue was dissolved in methylene chloride, and the solution was dropped into ethanol to obtain a powdery product (2.9 mg). When the dimethoxytrityl group was determined, the content of the limethoxytrityl group was found to be 0.401 mmol/g and the condensation yield was 97%.

The residue was dissolved in pyridine - triethylamine (3:1 v/v) (8 mL), the solution was stirred at room temperature for 2 hours, and the reaction solution was concentrated under a reduced pressure. The residue was dissolved in pyridine (10 mL), and the solution was dropped into ethanol (200 mL) under vigorous stirring, the formed precipitate <9> [see formula (5)] was removed by filtration, and the filtrate was concentrated under a reduced pressure. The residue was separated by reverse-phase column chromatography (eluent: 40% to 60% acetone - 0.05 M aqueous solution of TEAB) to obtain 0.2926 g of a dinucleotide derivative <8> (0.173 mmol, 58%). In this step, 0.526 g (0.05 mmol) of compound < 5 > was retained.

The Rf value of TLC using silica gel as the support and the data of 1H-n.m.r. of the compound <8> were in agreement with those of <8'> synthesized by the liquid phase method, and it was confirmed that <8> had the structure of <8'>.

The values of physical properties of compound <8> (B1 = B2 = N3-anisoyluracil-1-yl) were as follows (for this measurement, the diastereomer of 2'-O-tetrahydropyranyluridine with a high polarity was used).

¹H/n.m.r. (CDCl₃-TMS): δ1.21 (9H, t, J = 7.3 Hz, C₂ x 3), 1.27 - 1.75 (12H, m, C-C₃ x 6), 2.95 (6H, q, C-C₂ x 3), 3.41 - 3.68 (6H, m, 11-5',5' and O-C₂ x 2), 3.796, 3.80, 3.83 and 3.85 (12H, s x 4, OC₃ x 4), 4.16 - 5.75 (12H, m, H-5 x 2, 2' x 2, 3' x 2, 4' x 2, 5', 5" and -O-C -O x 2), 5.30 (1H, s, -OH), 6.05, 6.11, 6.15 and 6.27 (2H, d x 4, Hl' x 2), 6.83 - 7.92 (31H, m, phenyl proton x 29 and H6 x 2)

Rf values: 0.28 (acetone - water, 6:4 v/v), 0.65 (acetone - water, 7:3 v/v)

Example 3 Synthesis of the dinucleotide 3'-phosphodiester derivative < 8 > (B₁ = N⁻-anisoyluracil-1-yl, B₂ = N⁶-benzoyladenin-9-yl) using the acetylcellulose derivative < 4 >

According to the same procedures as described in Example 2, the compound < 4 > (1.65 mmol/g) (0.2424 g, 0.4 mmol) and triethylamine-³N-anisoyl-5'- O-dimethoxytrityl-2'- O'-tetrahydropyranyluridine-3,-(2-chlorophenyl)phosphate < 5 > (0.3170 g, 0.3 millimole) in pyridine (3 mL) as the solvent was reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.2726 g, 0.9 mmol) and 1-methylimidazole (0.14 mL, 1.8 mmol), and the reaction mixture was treated with acetic anhydride (2.5 mL) - pyridine (7.5 mL) (the condensation yield was 99%). Then, the reaction mixture was treated with a 2% solution of p-toluenesulfonic acid in chloroform - methanol (7:3 v/v), the reaction mixture was dissolved in chloroform, and the solution was dropped into ethanol to obtain 0.3680 g of the powdery compound <6> (0.255 mmol 85%, 0.693 mmol/g).

Compound <6> (0.3680 g) and triethylamine-N6-benzoyl-5'-O-dimethoxytrityl-2'-O-tetrahydropyranyladenosine-3,-(2-chlorophenyl)phosphate <5> (0.3359 g, 0.32 mmol) were reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.2907 g, 0.96 mmol) and 1-methylimidazole (0.15 mL, 1.92 mmol) in pyridine (3.2 mL) as the solvent (the condensation yield was 96%). The reaction mixture was treated with triethylamine-pyridine (1:3 v/v) (8 mL), and the acetylcellulose residue was precipitated by ethanol, retained by filtration and separated by reversed-phase silica gel column chromatography to obtain 0.2978 g of the dinucleotide derivative <8> (1.77 mmol, 59%). In this step, 0.0630 g (0.06 mmol) of the compound <5> (B2 = N6-benzoyladenin-9-yl) was obtained. It is to be noted that the Rf value of TLC and the data of 1H-n.m.r. of the compound <8> were identical with those of the compound <8'> synthesized by the liquid phase method, and it was confirmed that the compound <8> had the structure of the compound <8'>.

The values of physical properties of compound <8> (B1 = N3-anisoyluracil-1-yl, B2 = N6-benzoyladenin-9-yl) were as follows (the diastereomers of 2'-O-tetrahydropyranyluridine and 2'-O-tetrahydropyranyladenosine with a high polarity were used for the measurement).

¹H-n.m.r. (CDCl₃/TMS): δ1.19 - 1.66 (12H, m, C-C₂ x 6), 1.22 (9H, t, J = 7.3 Hz, C₃ x 3), 2.96 (6H, q, C-C₂ x 3), 3.04 - 3.78 (6H, m, H5', 5" and O-C₂ x 2), 3.75 (6H, s, OC₃), 3.83 (3H, s, OC₃), 4.28 - 5.84 (11H, m, H-5, 2' x 2, 3' x 2, 4' x 4, 5', 5" and O-C-O x 2), 5.38 (1H, s, -OH) 6.15, 6.17, 6.34 and 6.39 (2H, d x 4, H-1' x 2), 6.76 - 8.67 (33H, m, phenyl proton x 30, H-2.8 and H-6), 9.28 (1H, br s, NH)

Rf values: 0.30 (acetone - water, 6:4 v/v), 0.6 (acetone water, 7.3 v/v)

Example 4 Synthesis of the dinucleotide 3'-phosphodiester derivative < 8 > (B₁ = B₂ = N⁶-benzoyladenin-9-yl) using the acetylcellulose derivative < 4 >

According to the same procedures as described in Example 2, the Compound <4> (1.65 mmol/g) (0.6061 g, 1.00 mmol) and triethylamine-N⁶-benzoyl-5'-O- dimethoxytrityl-2'-O-tetrahydropyranyladenosine 3'-(2-chlorophenyl)phosphate (0.7872 g, 0.75 mmol) in pyridine (7.5 mL) as the solvent were reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.6814 g, 2.25 mmol) and 1-methylimidazole (0.36 mL, 4.5 mmol), and the reaction mixture was treated with acetic anhydride (6.25 mL) - pyridine (18.25 mL) (the condensation yield was 96%). The reaction mixture was treated with a 2% solution of p-toluenesulfonic acid in chloroform - methanol (7:3 v/v) and was dissolved in chloroform, and the solution was dropped into ethanol to obtain 0.9166 g of the powdery compound <6> (0.638 mmol, yield of 85%, 0.696 mmol/g). The compound <6> (0.9166 g) and triethylamine-N6-benzoyl-5'-O-dimethoxytrityl-2'-O-tetrahydropyranyladenosine 3,-(2-chlorophenyl)phosphate <5> (0.8370 g, 0.80 mmol) were reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.7269 g, 2.40 mmol) and 1-methylimidazole (0.38 mL, 4.8 mmol) in pyridine (8 mL) as the solvent (the conversion yield was 94%). The reaction mixture was treated with triethylamine - pyridine (1:3 v/v) (20 mL), and the acetylcellulose <9> was precipitated by ethanol, retained by filtration and separated by reversed-phase silica gel column chromatography to obtain 0.7701 g of the dinucleotide derivative <8> (0.459 mmol], yield of 61%). In this step, 0.1679 g (0.160 mmol) of the compound <5> (B2 = N6-benzoyladenin-9-yl) was recovered.

The Rf values and 1H n.m.r. data of compound <8> were identical to those of compound <8'> synthesized by the liquid phase method, and it was confirmed that compound <8> had the structure of compound <8'>.

The values of physical properties of compound <8> (B1 = B2 = N6-benzoyladenin-9-yl) were as follows (the diastereomer of 2'-O-tetrahydropyranyladenosine with a low B1 polarity and a high B2 polarity was used for the measurement).

1H-nmr (CDCl₃/TMS): δ1.22 - 1.62 (12H, m, C-CH₂ x 6), 1.25 (9H, t, J = 7.3 Hz, C 3 x 3), 2.60 - 3.85 (6H, m, H-5', 5" and -CC 2; x 2), 3.00 (6H, q, -CC 2; x 3), 3.74 and 3.75 (6H, sx 2, OC 3; x 2), 4.53 - 5.51 (10H, m, H-2' x 2, 3' x 2, 4' x 2,5',5" and OC -O x 2), 5.30 (1H, s, -OH), 6.26, 6.31, 6.36 and 6.39 (2H, dx 4, H-1' x 2), 6.74 - 8.79 (35H, m, H-2 x 2, 8 x 2 and phenyl proton x 31), 9.20 - 9.44 (2H, m, -NH x 2)

Rf values: 0.32, 0.29 (acetone - water, 6:4 v/v), 0.69, 0.66 (acetone - water, 7:3 v/v)

Example 5 Synthesis of the tetranucleotide derivative (B₁ = B₂ = B₃ = B₄ = N⁶-benzoyladenin-9-yl) < 10 > with a phosphodiester functional group at the 3'-end using 4-(2)hydroxyethylsulfonyl)dihydroxycinnamoylacetylcellulose < 4>

According to the same procedures as described above in the synthesis (i) of <8>, the compound <4> (1.65 mmol/g) (0.3182 g, 0.525 mmol) and a compound <8> (B1 = B2 = N6-benzoyladenin-9-yl) (0.5871 g, 0.35 mmol) in pyridine (10.5 mL) as the solvent were reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.3180 g, 1.05 mmol) and 1-methylimidazole (0.17 mL, 2.10 mmol), and the reaction mixture was treated with acetic anhydride (2.5 mL) - pyridine (7.5 mL) (the condensation yield was 100%). Then, the reaction mixture was treated with a 2% solution of toluenesulfonic acid in chloroform - methanol (7:3 v/v) and dissolved in chloroform, and the solution was dropped into ethanol to obtain 0.6248 g of a powdery acetylcellulose derivative having a dinucleotide supported thereon with a free carboxyl group at the 5'-end (0.288 mmol, yield of 82%, 0.462 mmol/g).

This powder (0.6248 g) and a compound <8> (B1 = B2 = N6-benzoyladenin-9-yl) (0.6760 g, 0.403 mmol) were reacted with 2,4,6-triisopropylbenzenesulfonyl chloride (0.3662 g, 1.21 mmol) and 1-methylimidazole (0.20 mL, 2.42 mmol) in pyridine (8 mL) as the solvent (the condensation yield was 90%).

Therein, the reaction mixture was treated with triethylamine - pyridine (1:3 v/v) (10.5 mL), and the acetylcellulose residue was precipitated by ethanol, retained by filtration, and separated by reversed-phase silica gel column chromatography to obtain 0.5095 g (0.174 mmol, yield 50%) of a tetranucleotide derivative. In this step, 0.1778 g (0.106 mmol) of compound < 8 > was obtained.

The values of physical properties of compound <10> (B1 = B2 = B3 = B4 = N6-benzoyladenin-9-yl) were as follows (the diastereomer of 2'-O-tetrahydropyranyladenosine with a high polarity was used for the measurement).

¹H-n.m.r. (CDCl₃/TMS): δ1.12 - 1.76 (24H, m, C-C₂ x 12), 1.23 (9H, t, J = 7.3 Hz, C₃ x 3), 2.90 - 3.56 (8H, m, O-CH₂- x 4), 2.97 (6H, q, C-CH₂ x 4), 3.68 - 3.92 (2H, m, H-5' and 5"), 3.72 (6H, s, OC₃ x 2), 4.12 - 5.72 (22H, m, H-2' x 4, 3' x 4, 4' x 4, 5' x 3, 5" x 3 and O-CH-O x 4). 5.30 (1H, s, -OH), 6.10 - 6.35 (4H, m, H - 1' x 4), 6.73 - 8.81(57H, m, H -2 x 4, 8 x 4 and phenyl proton x 49), 9.20 - 9.76 (4H, m, NH x 4)

Rf values: 0.10 (acetone - water, 6:4 v/v), 0.45 (acetone - water, 7:3 v/v)

Example 6 Synthesis of 5'-O-dimethoxytrityl-O6-diphenylcarbamoyl-N2-isobutyryl-2'-O- (tetrahydropyran-2-yl)guanosine-3'-succinate < 11 >

5'-O-Dimethoxytrityl-O⁶-diphenylcarbamoyl-N²-isobutyryl-2'-O-(tetrahydropyran-2-yl)guanosine (1.6670 g, 1.78 mmol) was dissolved in methylene chloride (8.9 mL), and succinic anhydride (0.3568 g, 3.57 mmol) and DMAP (0.4362 g, 3.57 mmol) were was added to the solution, and the mixture was stirred at room temperature for 30 minutes. Cold water (5 ml) was added to the mixture, and the mixture was stirred at room temperature for 30 minutes and extracted with methylene chloride (50 ml). The organic layer was washed with 0.1 M TEAB (20 ml x 3 times) and dried with anhydrous magnesium sulfate, and the magnesium sulfate was removed by filtration. The filtrate was subjected to distillation under a reduced pressure, and the residue was purified by silica gel column chromatography [2.5 cm in diameter x 10 cm in length, methanol - methylene chloride] to obtain 1.7370 g (the yield was 94%) of glassy 5'-O-dimethoxytrityl-O⁶-diphenylcarbamoyl-N²-isobutyryl-2'-O-(tetrahydropyran-2-yl)guanosine 3'-succinate <11>.

Rf value: 0.42 (CHCl₃ - MeOH, 9:1 v/v)

¹H-n.m.r. (CDCl3 - TMS): δ1.12 - 1.19 (6H, m, C(CH3)2), 1.24 - 1.70 (6H, m, C-CH2-C x 3), 2.51 - 2.84 (3H, m, C(CH3)2 and -CH2COOH), 2.96 - 3.23 (2H, m O-C2), 3.29 - 3.49 (2H, m, CH2COO-), 3.67 - 3.82 (2H, m, H - 5' and 5"), 3.75, 3.74 and 3.79 (9H, s x 3, OC3), 4.30 - 4.35 (1H, m, H - 4'), 4.68 - 4.74 (1H, m, O-CH-O), 5.25 (1H, dd, J2'3' = 4.88 Hz, Hz'), 5.50 - 5.56 (1H, m, H-3'), 6.27 (1H, d, J1'2' = 7.81 Hz, H - 1'), 6.77 - 7.76 (23H, m, phenyl proton x 23), 8.18 (1H, s, H - 8), 8.31 (1H, s, N² - H), 8.54 - 8.62 (1H, m, COO )

Elemental analysis values (C₅₇H₅₈N₆O₁₃H₂O):

Calculated values: C = 65.01, H = 5 74 N = 7.98

Values found: C = 64.74, H = 5.62, N = 7.92

Example 7 Synthesis of the acetylcellulose derivative < 12 > with guanosine 3'-succinate supported thereon with a free 5'-hydroxyl group

4-(2-Hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose <4> (1.65 mmol/g) (0.3636 g, 0.6 mmol) obtained in Example 1 and 5'-O-dimethoxytrityl-O6-diphenylcarbamoyl-N2isobutyryl-2'-O-(tetrahydropyran-2-yl)guanosine-3'-succinate <11> (0.4140 g, 0.4 mmol) obtained in Example 6 was subjected to azeotropic dehydration with pyridine (5 ml x 3 times), and the reaction mixture was dissolved in pyridine (6 ml). Then, 2,4,6-triisopropylbenzenesulfonyl chloride (0.3634 g, 1.2 mmol) and 1-methylimidazole (0.20 ml, 2.4 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. Cold water (2 ml) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was extracted with methylene chloride (60 ml) and washed with water (20 ml). The organic layer was concentrated and subjected to azeotropic dehydration with pyridine (5 ml x 3 times), acetic anhydride - pyridine (1:3 v/v) (15 ml) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 hours. The reaction liquid was concentrated, and a small amount of toluene was added, and azeotropic distillation was repeated until the pyridine odor disappeared. A small amount of the residue was dissolved in methylene chloride, and the solution was dropped into ethanol with vigorous stirring, and the dimethoxytrityl group was determined by using a white powder (4.3 mg) obtained by separating the precipitate formed (the amount carried thereon was 0.5 mmol/g, and the condensation yield was 98%).

The remaining residue was dissolved in chloroform-methanol (7:3 v/v) (5 ml), and with cooling to 0°C, a solution of p-toluenesulfonic acid monohydrate (0.3954 g) in chloroform-methanol (7:3 v/v) (5 ml) was added to the above solution, and the mixture was stirred for 15 minutes, neutralized by a 5% aqueous solution of sodium hydrogen carbonate and extracted with chloroform (100 ml). The organic layer was concentrated under a reduced pressure. The residue was dissolved in chloroform (10 ml), and the solution was added dropwise into ethanol (200 ml) with vigorous stirring. The precipitate formed was collected by filtration to obtain an acrylcellulose derivative < 12 > bearing a guanosine 3'-succinate with a free 5'-hydroxyl group in a yield of 66% (0.4511 g, 0.589 mmol/g).

Example 8 Synthesis of a hexanucleotide 3'-phosphodiester derivative < 13 > (m = 6, B₁ = B₃ = B₄ = UAN, B₂ = B₅ = B₆ = ABZ)

4-(2-Hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose < 4 > (1.65 mmol/g) (0.2727 g, 0.45 mmol) obtained in Example 4 and a dinucleotide 3'-phosphodiester derivative <8> (B₁ = UAN, B₂ = ABz) (0.5048 g, 0.3 mmol) was dissolved in pyridine (4.5 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (TPS) (0.2726 g, 0.9 mmol) and 1-methylimidazole (1-Meim) (0.144 mml, 1.8 mmol) were added to the solution to cause condensation. The reaction mixture was treated with acetic anhydride (2.5 mL) - pyridine (7.5 mL) (condensation yield was 94%). Then, the reaction mixture was treated with a 2% solution of p-toluenesulfonic acid monohydrate in chloroform - methanol (7:3 v/v) and dissolved in chloroform (10 mL). The solution was added dropwise into ethanol (200 mL) under vigorous stirring to obtain a powdery acetylcellulose derivative containing a dinucleotide having a free hydroxyl group at the 5'-end (n = 2, B1 = UAN, B2 = ABZ) in a yield of 72% (0.5090 g, 0.427 mmol/g).

Then, compound <14> (n=2, B1=UAN, B2=ABZ) (0.5090 g) and a dinucleotide 3'-phosphodiester derivative <8> (B1=B2=UAN) (0.5526 g, 0.326 mmol) were dissolved in pyridine (6.5 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.2962 g, 0.978 mmol) and 1-methylimidazole (0.156 mL, 1.96 mmol) were added to the solution to cause condensation (the condensation yield was 99%). The reaction mixture was dissolved in methylene chloride (10 mL), and the solution was added dropwise into ethanol (200 mL) with vigorous stirring to obtain a powdery acetylcellulose derivative <15> having a tetramer supported thereon with a yield of 61% (0.7307 g, 0.252 mmol/g).

It is worth mentioning that the filtrate was concentrated and the residue was purified by reverse phase silica gel column chromatography (2.5 cm i.d. x 10 cm in length, 40% to 60% acetone - 0.05 M TEAB aqueous solution) to retain 0.1251 g (0.072 mL) of <8> (B1 = B2 = UAN).

The powdered tetramer-supported acetylcellulose derivative <15> (0.7306 g) obtained by repeating the above-mentioned procedure was treated with acetic anhydride (2.5 ml) - pyridine (7.5 ml) and then treated with a 2% solution of p-toluenesulfonic acid monohydrate in chloroform - methanol (7:3 v/v). The reaction mixture was dissolved in chloroform (10 mL), and the solution was dropped into ethanol (200 mL) to obtain a powdery acetylcellulose derivative <16> supporting thereon a tetranucleotide having a free hydroxyl group at the 5'-end (n = 4, B1 = B3 = B - UAN, B2 = ABZ) in a yield of 47% (0.5173 g, 0.273 mmol/g).

Furthermore, <16> (n=4, B1=B3=B4=UAN, B2=ABZ) (0.5173 g) and <8> (B1=B2=ABZ) (0.3523 g, 0.21 mmol) were dissolved in pyridine (4.2 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.1908 g, 0.63 mmol) and 1-methylimidazole (0.1 mL, 1.26 mmol) were added to the solution to cause condensation (the condensation yield was 84%). Then, the reaction mixture was treated with a solution of triethylamine - pyridine (1:3 v/v) to precipitate the residual portion of acetylcellulose converted to a sulfonylalkene from ethanol. The precipitate was collected by filtration and separated by reverse-phase silica gel column chromatography to obtain a hexanucleotide 3'-phosphodiester derivative <13> (m = 6, B1 = B3 = B4 = UAN, B2 = B5 = B6 = ABZ) in a yield of 21% (0.2527 g). In this step, 0.1310 g (0.078 mmol) of a dinucleotide 3'-phosphodiester derivative <8> (B1 = B = ABZ) was retained.

The values of physical properties of the hexanucleotide 3'-phosphodiester derivative <13> (m = 6, B1 = B3 = B4 = UAN, B2 = B5 = B6 = ABZ) were as follows.

Rf values: 0.05 (acetone - water, 6:4 v/v), 0.27 (acetone - water, 7:3 v/v).

Example 9 i) Synthesis of undecamer (AAAAAAUUAUG) < 17 > using an acetylcellulose derivative as a carrier

A hexanucleotide derivative <13> (DMTr[AAUUAU]pOH) (0.4208 g, 0.1 mmol) having a phosphodiester functional group at the 5'-end and the acetylcellulose derivative <12> (0.4244 g, 0.25 mmol) having a guanosine 3'-succinate having a free 5'-hydroxyl group supported thereon obtained in Example 7 were subjected to azeotropic dehydration with pyridine (5 ml x 3 times), and the reaction mixture was dissolved in pyridine (5 ml).

2,4,6-Triisopropylbenzenesulfonyl chloride (0.1211 g, 0.4 mmol) and 1-methylimidazole (0.065 ml, 0.8 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. Then, cold water (2 ml) was added into the reaction mixture, and the mixture was stirred at room temperature for 30 minutes, extracted with methylene chloride (60 ml) and washed with water (20 ml). The organic layer was concentrated and subjected to azeotropic dehydration with pyridine (5 ml x 3 times), acetic anhydride - pyridine (1:3 v/v) (10 ml) was added to the residue, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated, and a small amount of toluene was added to the residue, and azeotropic distillation was repeated until the pyridine odor disappeared (the concentration yield was 98%, 0.117 mmol/g). The residue was dissolved in chloroform-methanol (7:3 v/v) (10 ml), and with cooling to 0 °C, a solution of p-toluenesulfonic acid monohydrate (0.3954 g) in chloroform-methanol (7:3 v/v) (10 ml) was added to the solution. The mixture was stirred for 15 minutes, neutralized with a 5% aqueous solution of sodium hydrogen carbonate and extracted with chloroform (100 ml), and the organic layer was concentrated under a reduced pressure. The residue was dissolved in chloroform (10 mL), and the solution was added dropwise into ethanol (200 mL) with vigorous stirring. The precipitate formed was collected by filtration to obtain an acetylcellulose derivative <18> having a heptamer with a hydroxyl group at the 5'-end supported thereon in a yield of 87% (0.7210 g, 0.121 mmol/g).

The thus obtained cellulose derivative <18> (0.7210 g, 0.087 mmol) and the tetranucleotide derivative <10> having a phosphodiester functional group at the 3'-end (DMTr[AAAA]pOH) (0.6424 g, 0.219 mmol) obtained in Example 5 were subjected to azeotropic dehydration with pyridine (5 mL x 3 times), and the residue was dissolved in pyridine (8.7 mL). 2,4,6-triisopropylbenzenesulfonyl chloride (0.1990 g, 0.657 mmol) and 1-methylimidazole (0.107 mL, 1.314 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. Then, cold water (2 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes, extracted with methylene chloride (100 mL) and washed with water (30 mL). The organic layer was concentrated under a reduced pressure, the residue was dissolved in methylene chloride (10 mL), and the solution was dropped into ethanol (200 mL) with vigorous stirring. The precipitate formed was collected by filtration to obtain an acetylcellulose derivative <19> having an undecamer supported thereon with a total yield of 69% (0.7911 g, 0.087 mmol/g) (the condensation yield was 96%). It is to be noted that 0.2180 g (0.074 mmol) of the tetranucleotide derivative <10> was retained from the filtrate by removing ethanol by distillation and performing purification by reversed-phase silica gel column chromatography.

ii) Removal of a protecting group from the undecamer (AAAAAAUUAUG)

The undecamer-supported acetylcellulose derivative <19> (44.8 mg, 3.97 mmol) was dissolved in a 0.5 M solution of 2-pyridinecarboxyaldoxime-1,1,3,3-tetramethylguanidine in pyridine-water (9:1 v/v) (0.6 mL), and the solution was allowed to stand at room temperature for 1 day. Ethanol (30 mL) was added to the reaction solution to precipitate the acetylcellulose derivative residue <9> from which the undecamer had been released, and centrifugal separation was carried out (3000 rpm, 4°C, 10 minutes), and the supernatant liquid was concentrated. Concentrated aqueous ammonia (28%) (20 mL) was added to the residue, and the mixture was covered, allowed to stand at 55°C for 6 hours, and concentrated under a reduced pressure. SEP-PAK (C-18) (Waters Co.) which was allowed to stand for more than 3 hours after injection of acetonitrile - water (9:1 v/v) (10 mL) was washed with 20 mL of 50 mM TEAB (prepared by diluting 1 M TEAB with pH 8.0), and the sample (residue) dissolved in 50 mM TEAB (20 mL) was injected into the washed SEP-PAK (C-18) (Waters Co.).

2-Pyridinecarboxyaldoxime and 1,1,3,3-tetramethylguanidine were eluted by 15% acetonitrile-50 mM TEAB (30 mL) and then by 18% acetonitrile-50 mM TEAB (10 mL), and the undecamer was eluted by 35% acetonitrile-50 mM TEAB (10 mL), and the eluate was freeze-dried. The residue was dissolved in water (1 mL), and the sample (0.1 mL) was purified by HPLC (M&S PACK C-18, 4.6 mm in inner diameter x 150 mm in length, 10% to 50% acetonitrile-0.1 M TEAA) (the results obtained are shown in Fig. 1). The eluate was confirmed by a UV monitor (280 nm), and the central part of the main peak was collected and concentrated under reduced pressure. Water (1 ml) was added to the concentrate, and distillation was carried out under a reduced pressure until the triethylamine disappeared. The residue was dissolved in an aqueous hydrochloric acid solution (5 ml) with a pH of 2.0, and the solution was allowed to stand at room temperature for 2 days. The solution was neutralized with dilute aqueous ammonia and washed with ethyl acetate (10 ml x 3 times), and the aqueous phase was concentrated under a reduced pressure. The sample (residue) was dissolved in water (200 µl) and the sample (180 µl) was purified by HPLC (M & S PACK C-18, 4.6 mm in inner diameter x 150 mm in length, 5% to 19% acetonitrile 0.1 M TEAA) (the results obtained are shown in Fig. 2). At this step, the eluate was confirmed by a UV monitor and the The middle part of each peak was collected and freeze-dried to obtain 1.51 OD of an undecamer (ApApApApApApUpUpApUpG) from which all protecting groups had been removed.

iii) Confirmation of the structure of the undecamer (ApApApApApApUpUpApUpG))

A 1 M ammonium acetate buffer (pH 5.3) (10 μl) and nuclease P1 (Yamasa Shoyu Co., 1 mg/5 μl) (3 μl) were added to an aqueous solution (86.32 μl) of the deprotected undecamer <17> (0.1 OD), and the mixture was allowed to stand at 37°C for 30 minutes.

The enzyme degradation product was separated by HPLC (M & S PACK C-18, 4.6 mm in inner diameter x 150 mm in length).

The undecamer was completely decomposed to obtain a decomposition product containing adenosine, guanosine 5-phosphate, uridine 5'-phosphate and adenosine 5'-phosphate in a ratio of 1.38:1.31:3.00:5.95.

It is said that nuclease P1 has the property of further decomposing adenosine 5'-phosphate into adenosine. The ratio determined in view of this fact, i.e. the ratio of (adenosine + adenosine 5'-phosphate) to uridine 5'-phosphate to guanosine 5'-phosphate is 7.00:2.87:1.25.

Example 10 Synthesis of the di-2'-deoxyribonucleotide-3'-phosphodiester derivative < 20 > (B₁ = B₂ = TBZ) supported on the acetylcellulose derivative < 4 >

A di-2'-deoxyribonucleotide-3'-phosphodiester <20> (B1 = B2 = TBZ) was synthesized from 4-(2-hydroxyethylsulfonyl)dihydrocinnamoylacetylcellulose <4> obtained in Example 1 according to the following reaction formula: Acetylcellulose Pyridine l-Meim Acetylcellulose reversed phase silica gel chromatography

The acetylcellulose derivative <4> (1.65 mmol/g) (0.3272 g, 0.53 mmol) obtained in Example 1 and triethylammonium N³-benzoyl-5'-O-dimethoxytritylthymidine-3'-(2-chlorophenyl)phosphate <21> (0.3766 g, 0.4 mmol) were dissolved in pyridine (3 ml), and the pyridine was removed by distillation under a reduced pressure. This procedure was repeated 3 times to remove water in the mixture by azeotropic Distillation. The residue was dissolved in pyridine (4 ml), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.3634 g, 1.2 mmol) and 1-methylimidazole (0.19 ml, 2.4 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (0.5 ml) was added to the reaction solution, and the mixture was stirred for 30 minutes, and the reaction solution was concentrated under a reduced pressure. Pyridine (3 ml x 3 times) was added and removed by distillation under a reduced pressure to remove water in the mixture by azeotropic dehydration. The residue was dissolved in pyridine (9 ml), and acetic anhydride (3 ml) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure. A small amount of toluene was added to the residue, and distillation was carried out under a reduced pressure until the pyridine odor disappeared. A very small amount of the acetyl cellulose derivative with <21> incorporated therein was dissolved in a small amount of methylene chloride, and the solution was dropped into ethanol, and the precipitate formed was collected by filtration and dried under a reduced pressure to obtain a powder (4.6 mg).

A 2% solution (1 ml) of p-toluenesulfonic acid in chloroform - methanol (7:3 v/v) was added to the powder and the mixture was allowed to stand at room temperature for 15 minutes to remove the dimethoxytrityl group and coloring was effected by 60% perchloric acid - methanol (3:2 v/v). By measuring the decadic absorbance at 498 nm (ε = 72000), the amount of < 21 > incorporated into the acetylcellulose derivative was determined. It was found that the amount of < 21 > incorporated was 0.604 mmol/g and the condensation yield was 100%.

The remaining acetyl cellulose derivative having < 21 > incorporated therein was dissolved in chloroform-methanol (7:3 v/v) (8 ml), and with cooling to 0 °C, a solution (4 ml) of p-toluenesulfonic acid monohydrate (0.3164 g) in chloroform-methanol (7.3 v/v) was added to the above solution, and the mixture was stirred for 15 minutes. Then, pyridine (1 ml) was added to the mixture, and the reaction solution was concentrated under a reduced pressure until the volume was reduced to about 1/2. The remaining solution was dropped into ethanol (200 ml) with vigorous stirring, and the precipitate formed was collected by filtration and dried under a reduced pressure to obtain a powdery acetyl cellulose derivative. < 22 > (B₁ = TBZ) with a thymidine-3'-phosphate derivative supported thereon having a free hydroxyl group at the 5'-position in a yield of 95% (0.5120 g, 0.378 mmol; 0.739 mmol/g).

<22> (B1 = TBZ) and <21> (B2 = TBZ) (0.449 g, 0.473 mmol) were added to pyridine (3 mL x 3 times), and water in the mixture was removed by azeotropic dehydration. The mixture was dissolved in pyridine (9.5 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.4298 g, 1.42 mmol) and 1-methylimidazole (0.225 mL, 2.84 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour (0.432 mmol/g, condensation yield = 99%). Cold water (1 mL) was added to the reaction solution, the mixture was stirred at room temperature for 30 minutes, and the reaction solution was concentrated under a reduced pressure.

Then, the residue was dissolved in triethylamine - pyridine (1:3 v/v) (16 mL), and the solution was stirred at room temperature for 2 hours. The residual portion of the acetylcellulose derivative <9> converted to the sulfonylalkene was precipitated by ethanol (100 mL) and removed by filtration. The filtrate was concentrated under a reduced pressure and separated by reverse phase silica gel chromatography (10% to 40% acetone - 0.05 M TEAB system) to obtain <20> (B1 = B2 = TBZ) with a yield of 63% (0.3710 g, 0.254 mmol). In this step, 0.789 g (0.083 mmol) of <21> (B = TBZ) was recovered.

The physical properties of < 20 > (Bi = B₂ = TBZ) were as follows.

Rf value: 0.29 (acetone - water, 6:4 v/v)

¹H-n.m.r. (CDCl3 - TMS): δ1.24 (9H, t, J = 7.3 Hz, [C-C3 in N-C2H5 group] x 3), 1.27 (3H, s, CH3-5), 1.35 (3H, s, CH3-5), 2.05 - 2.70 (4H, m, H - 2' x 2 and H - 2" x 2), 2.97 (6H, q, C-C2-N x 3), 3.78 (6H, s, OCH3 x 2), 3.30 - 4.50 (6H, m, H - 4' x 2, H - 5' x 2 and H - 5" x 2), 4.95 - 5.40 (2H, m, H - 3' x 2), 6.29 - 6.45 (2H, m, H - 1' x 2) and 6.30 - 8.60 (33H, m, phenyl proton x 31 and H - 6 x 2)

Example 11 Synthesis of the di-2'-deoxyribonucleotide-3'-phosphodiester derivative < 20 > B₁ = B₂ = AAca) using the acetylcellulose derivative < 4 > as a carrier

According to the reaction formula described in Example 10, a di-2'-deoxyribonucleotide-3-phosphodiester derivative <20> (B1 = B2 = AACa) was synthesized from the acetylcellulose derivative <4>.

More specifically, in the same manner as described in Example 10, <4> (1.65 mmol/g) (0.3272 g, 0.53 mmol) and triethylammonium 5'-O-dimethoxytrityl-N⁶-(dimethylamino)ethylene-2'-deoxyadenosine 3'-(2-chlorophenyl)phosphate <21> (0.3882 g, 0.4 mmol) were dissolved in pyridine (4 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.3634 g, 1.2 mmol) and 1-methylimidazole (0.19 mL, 2.4 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (1 mL) was added to stop the reaction, and the reaction solution was concentrated under a reduced pressure and treated with acetic anhydride - pyridine (1:3 v/v) (12 mL) (0.590 mmol/g, condensation yield = 99%). Then, the reaction solution was treated with a solution (12 mL) of p-toluenesulfonic acid monohydrate (0.3164 g) in chloroform-methanol (7:3 v/v) and added dropwise into ethanol (200 mL) under vigorous stirring to obtain powdery <22>(B1-AAca) with a yield of 89% (0.4970 g, 0.357 mmol; 0.717 mmol/g).

<22> (B₁ = AAca) and <21> (B₂ = AAca) (0.4325 g, 0.446 mmol) were dissolved in pyridine (9 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.3377 g, 1.34 mmol) and 1-methylimidazole (0.21 mL, 2.68 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (1 mL) was added to stop the reaction, and the reaction solution was concentrated under reduced pressure so that the volume was reduced to about 1/2, and the concentrate was dropped into ethanol (200 mL) with vigorous stirring to obtain a powdery dinucleotide-supported acetylcellulose derivative <23> in a yield of 0.6900 g, 0.283 mmol (0.410 mmol/g, condensation yield = 97%).

The acetylcellulose derivative residue <9> converted to a sulfonylalkene by treatment with triethylamine-pyridine (1.3 v/v) (16 mL) was precipitated from ethanol (100 mL), retained by filtration and separated by reversed phase silica gel chromatography to obtain <20> (B1 = B2 = AAca) in a yield of 63% (0.3710 g, 0.254 mmol). In this step, 0.0789 g (0.083 mmol) of <21> (B = AAca) was recovered.

The physical properties of < 20 > (B₁ = B₂ = AAca) are as follows.

Rf value: 0.34 (acetone - water, 6:4 v/v)

¹H-n.m.r. (CDCl3 - TMS): δ1.27 (9H, t, J = 7.3 Hz, [C-C3 in N-ethyl group] x 3), 2.10 (6H, s, C(C3) of N(C3)2 x 2), 2.85 - 2.90 (4H, m, H - 2' x 2 and H - 2" x 2), 2.90 (6H, q, C-C2-N x 3), 3.13 (12H, m, N(CH3)2 x 2), 3.34 (4H, m, H - 5' x 2 and H - 5" x 2), 3.72 (6H, s, O-CH3 x 2), 4.40 (2H, m, H - 4' x 2), 5.40 - 5.55 (2H, m, H - 3' x 2), 6.36 - 6.50 (2H, m, H - 1' x 2), 6.70 - 7.50 (21H, m, phenyl proton x 21), 8.00 (2H, s, H - 2 x 2) and 8.55 (2H, s, H - 8 x 2)

Example 12 Synthesis of the di-2'-deoxyribonucleotide-3'-phosphodiester derivative < 20 > (B₁ = B₂ = GDpc,iBu) using the acetylcellulose derivative < 4 > as a carrier

According to the reaction formula described in Example 10, a di-2'-deoxyribonucleotide-3'-phosphodiester derivative < 20 > (B₁ = B₂ = GDpc,iBu) was synthesized from the acetylcellulose < 4 >

More specifically, in the same manner as described in Example 10, <4> (1.65 mmol/g) (0.2424 g, 0.4 mmol) and triethylammonium 5'-O-dimethoxytrityl-N⁶-diphenylcarbamoyl-N²-isobutyryl-2-deoxyguanosine 3'-(2-chlorophenyl)phosphate <21> (B₁ = GDpc,iBu) (0.3375 g, 0.3 mmol) were dissolved in pyridine (3 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.2726 g, 0.9 mmol) and 1-methylimidazole (0.144 mL, 1.8 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. Cold water (1 mL) was added to stop the reaction, and the reaction solution was concentrated under a reduced pressure and treated with acetic anhydride - pyridine (1:3 v/v) (10 mL) (0.533 mmol/g, condensation yield = 98%). Then, the reaction solution was treated with p-toluenesulfonic acid monohydrate (0.3164 g) and chloroform - methanol (7:3 v/v) (12 mL) and added dropwise into ethanol (200 mL) under vigorous stirring to obtain the powder <22> (B1 = GDpc,iBu) with a yield of 88% (0.4150 g, 0.264 mmol; 0.635 mmol/g).

<22> (B₁ = GDpc,iBu) and <21> (B₂ = GDpc,iBu) (0.3708 g, 0.33 mmol) were dissolved in pyridine (6.6 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.2999 g, 0.99 mmol) and 1-methylimidazole (0.16 mL, 1.98 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour.

Cold water (1 mL) was added to stop the reaction, and the reaction solution was concentrated under a reduced pressure (0.356 mmol/g, condensation yield = 95%). The concentrate was treated with triethylamine - pyridine (1:3 v/v) (8 mL), and the acetylcellulose derivative residue <9> converted to a sulfonylalkene was precipitated from ethanol (100 mL), retained by filtration and separated by reverse-phase silica gel column chromatography to obtain <20> (B1 = B2 = GDpc,iBu) with a yield of 51% (0.2797 g, 0.153 mmol). In this step, < 21 > (B = GDpc,iBu) was obtained in an amount of 0.0496 g (0.0441 mmol).

The physical properties of < 20 > (B₁ = B₂ = GDpc,iBu) are as follows.

Rf value: 0.17 (acetone - water, 6:4 v/v)

¹H-n.m.r. (CDCl3 - TMS): δ1.05 - 1.25 (21H, m [C-C3 in N-ethyl group] x 3 and CH(C3)2 x 2), 2.50 - 3.45 (14H, m, C-C2-N x 3, CH(CH3)2 x 2, H - 2' x 2, H - 2" x 2, H - 5' x 2 and H - 5" x 2), 3.70 (6H, s, OCH3 x 2), 4.50 - 4.65 (2H, m, H - 4' x 2), 5.30 - 5.55 (2H, m, H - 3' x 2), 6.30 - 6.45 (2H, m, H - 1' x 2), 6.66 - 7.64 (41H, m, phenyl proton x 41), 8.05 and 8.07 (2H, s x 2, H - 8 x 2) and 8.55 and 8.80 (2H, s x 2 N CO x 2)

Example 13 Synthesis of the tri-2'-deoxyribonucleotide-3'-phosphodiester derivative <24> (B1 = CAn, B2 = B3 = TBZ) using the acetylcellulose derivative <4> as the carrier

A tri-2'-deoxyribonucleotide-3'-phosphodiester derivative < 24 > was synthesized from the acetylcellulose derivative < 4 > according to the following reaction formula: Acetylcellulose 1-Meim Et₃N-pyridine reversed phase silica gel column chromatography

The acetylcellulose derivative <4> (1.63 mmol/g) (0.8650 g, 1.39 mmol) obtained in Example 1 and N4-anisoyl-5'-O-dimethoxytrityl-2'-deoxycytidine-3'-(2-chlorophenyl)phosphate <21> (0.9550 g, 1 mmol) were subjected to azeotropic dehydration with pyridine (5 ml x 3) to remove water from the system, and the mixture was dissolved in pyridine (10 ml). Then, 2,4,6-triisopropylbenzenesulfonyl chloride (0.9086 g, 3 mmol) and 1-methylimidazole (0.48 ml, 6 mmol) were added to the solution, and the mixture was stirred at room temperature for 4 hours. Cold water (0.5 ml) was added to the reaction solution to stop the reaction, and the reaction mixture was concentrated under a reduced pressure, water was removed from the reaction mixture by azeotropic dehydration with pyridine (5 ml x 3 times), the residue was dissolved in pyridine (7.5 ml), Acetic anhydride (2.5 mL) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, and a small amount of toluene was added to the concentrate, and distillation was carried out under a reduced pressure until the pyridine odor was no longer perceived (0.554 mmol/g, condensation yield = 82%).

Then, the residue was dissolved in a solution (20 ml) of p-toluenesulfonic acid monohydrate (0.5412 g) in chloroform - methanol (7:3 vv/v), and the solution was stirred at 0 °C for 15 minutes, neutralized with pyridine and concentrated under a reduced pressure. The residue was dissolved in methylene chloride (10 ml), and the solution was added dropwise into ethanol (100 ml) with vigorous stirring. The precipitate formed was collected by filtration and dried under a reduced pressure to obtain powdery <25> (B1 = CAn) with a yield of 64% (0.9624 g, 0.64 mmol; 0.665 mmol/g).

Then, <25> (B1 = CAn) and <20> (B1 = B2 = TBZ) (0.7565 g, 0.518 mmol) were subjected to azeotropic dehydration with pyridine (5 mL x 3 times) to remove water in the system, and the mixture was dissolved in pyridine (10 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.4840 g, 1.60 mmol) and 1-methylimidazole (0.26 mL, 3.2 mmol) were added to the solution. The mixture was stirred at room temperature for 4 hours. Cold water (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes and concentrated under a reduced pressure to obtain <26>.

The residue was dissolved in triethylamine-pyridine (1:3 v/v) (20 mL), and the solution was stirred at room temperature for 2 hours and concentrated under a reduced pressure. The residue was dissolved in methylene chloride (10 mL), and the solution was added dropwise into ethanol (100 mL) with vigorous stirring to precipitate the acetylcellulose derivative residue <9> converted into a sulfonylalkene. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure and separated by reverse-phase silica gel column chromatography (10% to 40% acetone - 0.05 M TEAB system) to obtain <24> (B1 = (CAn, B2 = B3 = TBZ) in 47% yield (0.5180 g, 0.25 mmol).

The physical properties of non < 24 > (B₁ = CAn, B₂ = B₃ = TBZ) are as follows.

Rf values: 0.19 (acetone - water, 6:4 v/v), 0.43 (acetone - water, 7:3 v/v)

Example 14 Synthesis of the tetra-2'-deoxyribonucleotide-3'-phosphodiester derivative <27> (B1 = B2 = AAca, B3 = B4 = GDpc,iBu) using the acetylcellulose derivative <4> as the carrier

A tetra-2'-deoxyribonucleotide-3'-phosphodiester derivative <27> (B1 = B2 = AAca, B3 = B4 = GDpc,iBu) was synthesized from the acetylcellulose derivative according to the following reaction formula: 1-Meim Pyridine Acetylcellulose Et₃N-pyridine Reversed phase silica gel column chromatography

The acetylcellulose derivative <4> (1.63 mmol/g) (0.3750 g, 0.604 mmol) obtained in Example 1 and <20> (B1 = B2 = AAca) (0.6140 g, 0.405 mmol) obtained in Example 11 were subjected to azeotropic dehydration with pyridine (5 ml x 3 times) to remove water in the mixture, and the mixture was dissolved in pyridine (10 ml). Then, 2,4,6-triisopropylbenzenesulfonyl chloride (0.3660 g, 1.21 mmol) and 1-methylimidazole (0.20 ml, 2.48 mmol) were added to the solution, and the mixture was stirred at room temperature for 4 hours. Cold water (0.5 mL) was added to the reaction solution to stop the reaction, the reaction solution was concentrated under a reduced pressure, and the residue was subjected to azeotropic dehydration with pyridine (5 mL x 3 times) to remove water in the mixture. The residue was dissolved in pyridine (7.5 mL), and acetic anhydride (2.5 mL) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, and a small amount of toluene was added to the concentrate, and distillation was repeated under a reduced pressure until the pyridine odor disappeared (0.345 mmol/g, condensation yield = 94%).

The residue was dissolved in a solution (20 ml) of p-toluenesulfonic acid monohydrate (0.5412 g) in chloroform - methanol (7:3 v/v), and the solution was stirred at 0 °C for 15 minutes, neutralized with pyridine and concentrated under a reduced pressure. The residue was dissolved in methylene chloride (10 ml), and the solution was added dropwise into ethanol (100 ml) with vigorous stirring. The precipitate formed was collected by filtration and dried under a reduced pressure to obtain powdery <28> (B₁=B₂=AACa) with a yield of 76% (0.7720 g, 0.306 mmol; 0.397 mmol/g).

Then, <28> (B1 = B2 = AACa) and <20> (B1 = B2 = GDpc,iBu) (0.6710 g, 0.36 mmol) obtained in Example 12 were subjected to azeotropic dehydration with pyridine (5 ml x 3 times) to remove water in the mixture, and the mixture was dissolved in pyridine (10 ml), and 2,4,5-triisopropylbenzenesulfonyl chloride (0.3336 g, 1.1 mmol) and 1-methylimidazole (0.15 ml, 2.21 mmol) were added to the solution. The mixture was stirred at room temperature for 4 hours, and cold water (0.5 ml) was added to the reaction solution. The mixture was stirred at room temperature for 30 minutes and concentrated under a reduced pressure.

The residue was dissolved in triethylamine - pyridine (1:3 v/v) (20 mL), the solution was stirred at room temperature for 2 hours and concentrated under a reduced pressure. The residue was dissolved in methylene chloride (10 mL), and the solution was added dropwise into ethanol (100 mL) with vigorous stirring to precipitate the residue of acetylcellulose derivative <9> converted into a sulfonylalkene, which was removed by filtration. The filtrate was concentrated under reduced pressure and separated by reversed-phase silica gel column chromatography (10% to 40% acetone - 0.05 M TEAB system) to afford <27> (B1 = B2 = AACa, B3 = B4 = GDpc,iBu) in 69% yield (0.6000 g, 0.21 mmol).

The value of physical properties of <27> (B1 = B2 = AAca, B3 = B4 = GDpc,iBu) is as follows.

Rf value: 0.17 (acetone - water, 7:3 v/v)

Example 15 Synthesis of octa-2'-deoxyribonucleotide < 29 > (GpGpApApTpTpCpC) using the acetylcellulose derivative < 4 >

According to the following reaction formula, an acetylcellulose derivative <30> having 2'-deoxycytidine-3'-succinate supported thereon was synthesized from acetylcellulose <4>, an acetylcellulose derivative <31> having octa-2'-deoxyribojinucleotide supported thereon is synthesized from <30>, and the protecting groups were removed from <31>, and purification was carried out to obtain octa-2'-deoxyribonucleotide <29> (GpGpApApTpTpCpC): 1-Meim Pyridine Acetylcellulose Acetylcellulose reversed phase HPLC

i) Synthesis of the acetylcellulose derivative < 30 > with 2'-deoxycytidine-3'-succinate supported thereon

The acetylcellulose derivative <4> (1.63 mmol/g) (0.9663 g, 1.58 mmol) and N4-anisoyl-3'-O-(3-carboxy)propionyl-5'-O-dimethoxytrityl-2'-deoxycytidine <32> (0.802 g, 1.05 mmol) were subjected to azeotropic dehydration with pyridine (5 ml x 3 times), and the mixture was dissolved in pyridine (10.5 ml), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.9450 g, 3.15 mmol) and 1-methylimidazole (0.53 ml, 6.3 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. Cold water (1 ml) was added to the reaction solution, and the mixture was stirred for 30 minutes, methylene chloride (100 ml) was added, and the mixture was washed with water (40 ml). The organic layer was concentrated under a reduced pressure, and the concentrate was subjected to azeotropic dehydration with pyridine (5 ml x 3 times). Acetic anhydride - pyridine (1:3 v/v) (20 ml) was added to the residue, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, and a small amount of toluene was added to the residue, and azeotropic distillation was carried out until the pyridine odor disappeared (0.5 mmol/g, condensation yield = 98%). The residue was dissolved in chloroform-methanol (7:3 v/v) (10 ml), and the solution was cooled to 0 °C, and a solution (10 ml) of p-toluenesulfonic acid moiety hydrate (0.5412 g) in chloroform-methanol (7:3 v/v) was added to the above solution. The mixture was stirred for 15 minutes and neutralized with pyridine, and the reaction solution was concentrated under a reduced pressure. The residue was dissolved in methylene chloride (20 ml), and the solution was added dropwise into ethanol (200 ml) with vigorous stirring. The precipitate formed was collected by filtration and dried under reduced pressure to obtain powdered <30> in 84% yield (1.3190 g, 0.877 mmol; 0.665 mmol/g).

ii) Synthesis of the acetylcellulose derivative < 31 > with octa-2'-deoxyribonucleotide supported thereon

<24> (B1 = CAn, B2 = B3 = TBZ) (0.2735 g, 0.132 mmol) obtained in Example 13 and <30> (0.4910 g, 0.33 mmol) obtained in i) above were subjected to azeotropic dehydration with pyridine (5 ml x 3 times) to remove water from the mixture, and the mixture was dissolved in pyridine (3.3 ml), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.1599 g, 0.528 mmol) and 1-methylimidazole (0.09 ml, 1.06 mmol) were added to the solution. The mixture was stirred at room temperature for 2 hours, and cold water (0.5 ml) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes and concentrated under a reduced pressure. The residue was subjected to azeotropic dehydration with pyridine (5 ml x 3 times) to remove water from the mixture. The residue was dissolved in pyridine (15 ml), and acetic anhydride (5 ml) was added to the solution, and the mixture was stirred at room temperature for 5 hours. The reaction solution was concentrated under a reduced pressure, and a small amount of toluene was added to the concentrate, and distillation under a reduced pressure was carried out until the pyridine odor disappeared (0.163 mmol/g, condensation yield = 93%). Then the residue was dissolved in chloroform-methanol (10 ml), and the solution was cooled to 0ºC, and a solution (10 ml) of p-toluenesulfonic acid monohydrate (0.5412 g) in chloroform-methanol (7.3 v/v) was added to the above solution was added. The mixture was stirred for 15 minutes and neutralized with pyridine, and the reaction solution was concentrated under a reduced pressure. The residue was dissolved in methylene chloride (10 ml), and the solution was added dropwise into ethanol with vigorous stirring. The precipitate formed was collected by filtration and dried under a reduced pressure to obtain powdery < 33 > in a yield of 86% (0.6685 g, 0.115 mmol; 0.171 mmol/g).

<33> (0.445 g, 0.076 mmol) and <27> (B5 = B6 = AAca, B7 = B8 = GDpc,iBu) (0.4343 g, 0.152 mmol) obtained in Example 14 were subjected to azeotropic dehydration with pyridine (5 ml x 3 times), and the mixture was dissolved in pyridine (3.4 ml), and 2,4,6-triisopropylbenzenesulfonyl chloride (0.1726 g, 0.57 mmol) and 1-methylimidazole (0.09 ml, 1.14 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. Cold water (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes and concentrated under a reduced pressure (condensation yield = 80.43%). The residue was dissolved in methylene chloride (10 mL), and the solution was added dropwise into ethanol (100 mL) with vigorous stirring. The precipitate formed was collected by filtration and dried under a reduced pressure to obtain powdery <31> with a yield of 91% (0.6120 g, 0.069 mmol; 0.113 mmol/g).

iii) Removal of protecting groups from the octamer < 31 > and purification

The octamer <31> (30 mg, 3.39 mmol) obtained in ii) above was dissolved in a 0.5 M solution of N¹,N¹,N³,N³-tetramethylguanidine-(E)-2-pyridinealdeoximate in pyridine-water (9:1 v/v) (0.6 ml), and the solution was allowed to stand at room temperature for one day. Ethanol (30 ml) was added to the reaction solution to precipitate the residual portion (9) of the acetylcellulose derivative, which was removed by filtration. The filtrate was concentrated under a reduced pressure. Concentrated aqueous ammonia (28%) (20 ml) was added to the residue, and the mixture was covered and allowed to stand at 55 °C for 6 hours, followed by concentration under a reduced pressure. The octamer residue dissolved in a 50 mM aqueous solution of TEAB (20 mL) was injected into SEP-PAK (C-18) (sold by Waters Co.) which had been allowed to stand for more than 3 hours after an injection of acetonitrile-water (9:1 v/v) (10 mL) and then washed with a 50 mM aqueous solution of TEAB (20 mL). Then, N¹,N¹,N³,N³-tetramethylguanidine-(E)-2-pyridine alde oximate was added at 5% Acetonitrile-0.05 mM TEAB aqueous solution (20 mL), and the octamer was then eluted with 10% acetonitrile-0.05 mM TEAB aqueous solution (20 mL) or 15% acetonitrile-0.05 M TEAB aqueous solution (10 mL). The eluate was concentrated under a reduced pressure, and the residue was dissolved in water (1 mL), and the sample (0.63 mL) was collected, and the octamer <34> having a dimethoxytrityl group at the 5'-position was purified by HPLC. The middle portion of the main peak was collected and concentrated under a reduced pressure. Water (1 mL) was added to the residue, and distillation was carried out under a reduced pressure until the triethylamine odor disappeared. Then, an 80% aqueous solution of acetic acid (1.31 mL) was added to 1/2 of the collected sample, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under a reduced pressure, and a small amount of water was added to the residue, and the acetic acid was removed by azeotropic distillation. The residue was dissolved in water (1 mL), and the sample (0.46 mL) was purified by HPLC [LiChrosorb RP-18, 4 mm in inner diameter x 250 mm in length, 10% acetonitrile - 0.1 M TEAA aqueous solution] (the results obtained are shown in Fig. 3). The middle portion of the main peak was collected, and the eluate was concentrated under a reduced pressure to 1.95 OD of the octamer <29> from which all the protecting groups had been removed (the results are shown in Fig. 4).

iv) Confirmation of the structure of the octamer < 29 > (GpGpApApTpTpCpC)

The octamer <29> (0.6 OD) was dissolved in 0.05 M Tris buffer (pH 8.48) (200 µl), and snake venom phosphodiesterase (20 µg) was added to the solution, and the mixture was allowed to stand at 37°C for 2 hours. Then, alkaline phosphatase (50 µg) was added to the mixture, and the mixture was allowed to stand at 25°C for 1 hour. The enzyme decomposition product was separated by HPLC [Lichrosorb RP-18, 4 mm in inner diameter x 250 mm in length, 10% acetonitrile - 0.1 M sodium phosphate aqueous solution (pH 4.2)], and it was confirmed that <29> was completely decomposed into the respective 2'-deoxynucleotides.

INDUSTRIAL APPLICABILITY

With the rapid progress in biochemistry or genetic engineering, especially with the development of recombinant DNA technology, the need for synthetic oligonucleotides has increased, and the present invention is useful for meeting this need.

Synthetic DNA is used for the synthesis of structural genes such as human interferons and human growth hormones, and further, the use of synthetic DNA decamers via eicosamers for the preparation of primers for determination of sequences, linkers for cloning, probes for selection and confirmation of target genes, agents for direct diagnosis (genetic diagnosis) of diseases transmitted by microorganisms, and drugs such as anti-virus agents has considerably increased in the medical and therapeutic fields and the like. Primers, linkers and probes have specific sequences, and large amounts of highly pure DNA oligomers are necessary for their preparation. The process of the present invention, which is simple and suitable for this synthesis in large quantities, makes a great contribution to their preparation.

In the protein biosynthesis system, RNA is located at an intermediate position between DNA and a protein and plays an important role. However, there are many unknown points, and in order to clarify the functions, it is desirable to obtain RNA with an optional sequence in large quantities. According to the method using enzymes, RNA is obtained only in a small amount, and the desired sequence is limited. Accordingly, the chemical synthesis method is very advantageous. However, according to the conventional liquid-phase phosphotriester method, it is very difficult to obtain high-purity RNA oligomers such as decamers in large quantities. Furthermore, in the solid-phase phosphotriester method, a general procedure for synthesizing RNA oligomers as in the synthesis of DNA oligomers has not yet been provided.

The process of the present invention is very valuable as the process for easily and in large quantities producing DNA oligomers and RNA oligomers, the synthesis of which is extremely laborious and cumbersome.

Claims (5)

1. A process for the synthesis of a chain-extended oligonucleotide, which comprises condensing a mononucleotide, an oligonucleotide, a mononucleotide succinate or an oligonucleotide succinate in a homogeneous system in a solvent, in which only the terminal 5'-hydroxyl group is not blocked and the other functional groups are blocked by a plurality of low molecular weight protective groups and a protective group containing a hydroxyl group, with a mononucleotide or oligonucleotide in which the functional groups except for the terminal phosphoric acid group are blocked by low molecular weight protective groups, characterized in that the protective group containing the hydroxyl group is a polysaccharide derivative which is controllably soluble in the solvent, wherein the compound which forms the protective group from the polysaccharide derivative is formed by bonding a spacer and a functional group to a polysaccharide derivative is formed. 2. A process for synthesizing an oligonucleotide according to claim 1, wherein the compound which forms the protective group of the polysaccharide derivative is represented by the following general formula (I): where C�6H�7O₂ is an anhydrous glucose residue, R is n is an integer between 10 and 2,000, x is a number between 0.4 and 0.8 and y is a number between 1.0 and 2.0. 3. A process for synthesizing an oligonucleotide according to claim 1 or claim 2, wherein the polysaccharide derivative in the compound forming the protecting group of the polysaccharide derivative is acetylcellulose. 4. A compound for forming a protecting group of a polysaccharide derivative, which is represented by the following general formula (I): where C�6H�7O₂ is an anhydrous glucose residue, R is n is an integer between 10 and 2,000, x is a number between 0.4 and 0.8 and y is a number between 1.0 and 2.0. 5. A compound according to claim 4, wherein R in formula (I) CH3 -. is.